The goal of this application is to address critical gaps in our understanding of the Non-homologous end joining (NHEJ) DNA repair pathway using new single molecule methods. NHEJ is the main pathway for repair of DNA double strand breaks (DSBs), the most cytotoxic form of DNA damage, resulting from Ionizing Radiation (IR) and chemotherapeutics. Mutations in NHEJ proteins are associated with genomic instability, IR sensitivity and severe combined immunodeficiency (SCID). Consequently, the targeted inhibition of the NHEJ pathway is of importance for sensitization of cancer cells in IR therapy. The mechanisms that control NHEJ play a key role in development and in response to cancer therapy, but the current state of knowledge regarding the NHEJ repair process is limited. We especially know very little about the physical nature of the NHEJ complex and how it is assembled, as common biochemical, structural and cell biology methods are limited in their capacities to resolve this information. Without this level of understanding, insights into NHEJ mutations that cause IR sensitivity and immunodeficiency and strategies for inhibiting NHEJ in neoplastic cells remain stagnant. In this application we utilize innovative single-molecule methods to define the NHEJ repair process. We use an array of new single-molecule biochemical methods to define the two critical steps of NHEJ: Assembly of NHEJ complex on DNA ends (Aim-1) and synapsis of DNA ends (Aim-2). Within these aims we will determine the specific functional organization of NHEJ complex components, their dependence on DNA end chemistry, and the consequence of their clinical and structural mutations. In Aim-3 we study NHEJ in cells. We will define the nanoscale architecture of NHEJ complexes in cells and determine their association with cellular DNA damage response (DDR) factors and characterize how these are modulated in different types of DSB lesions. Finally, we will determine the consequence of clinical and structural mutations in NHEJ proteins and the effect of NHEJ inhibitors on the organization of NHEJ complexes in cells. Our results will provide a platform for addressing novel hypotheses with cutting-edge single-molecule technology, with enormous potential for advancing the field of DNA damage research.